Development of procedures for generating monoclonal antibodies against specific cells is described for instance in Kohler, G. and Milstein, C. (1975) "Continuous cultures of fused cells secreting antibody of predefined specificity", Nature 256, 495-497. These procedures have underlined the need to develop new and improved immunological labeling techniques to detect and separate specific cells. An approach initiated by the present inventor in collaboration with Dr. A. Rembaum and S.P.S. Yen and described in Molday, R.S., Yen, S.P.S. and Rembaum, A. (1977) "Application of magnetic microspheres in labeling and separation of cells, "Nature 268, 437-438 involved the synthesis of magnetic microspheres by cobalt .gamma.-irradiation of iron oxide colloidal particles in the presence of hydrophilic and hydrophobic methacrylate monomers. These microspheres were coupled to immunoglobulin and used to label and separate cells by magnetic means. These magnetic reagents, however, were limited in application due to difficulties in synthesis and purification of the microspheres and, more important, susceptability to aggregation and nonspecific binding to certain types of cells. Kronick, P. L., Campbell, G., Joseph, K. (1978) "Magnetic microspheres prepared by redox polymerization used in a cell separation based on gangliosides", Science 200, 1074-1076 prepared similar magnetic polymeric particles, but these also appeared under the electron microscope as aggregated material on cell surfaces. Albumincoated microspheres have also been prepared for use as drug-carriers, Widder, K., Flouret, G. and Senyei, A. (1979) Magnetic microspheres: "Synthesis of a novel parenteral drug carrier", J. Pharm. Sci. 68, 79-82, but these reagents are relatively large in size, approximately 1 micron (10.sup.4 .ANG.) in diameter and, therefore, are limited as general reagents for cell labeling.
U.S. Pat. No. 3,970,518 of Giaever relates to the magnetic separation of biological particles such as cells, bacteria or viruses and makes use of magnetic particles coated with a layer of antibodies to the particles to be separated. The antibody coated magnetic particles contact a mixed population including the particles to be separated. The particles to be separated attach to the antibodies present on the magnetic particles, the magnetic particles are magnetically separated and the separated particles are subjected to a cleaving reaction to separate the required biological particles from the antibody-coated magnetic particles. The magnetic particles used can be ferromagnetic, ferrimagnetic or superparamagnetic. Suitable magnetic materials include oxides such as, for example, ferrites, perovskites, chromites and magnetoplumbites. The particles can range in size from colloidal to about 10 microns.
U.S. Pat. No. 4,018,886 of Giaever relates to a diagnostic method for determining the presence or absence of select proteins in low concentration in a liquid sample. A plurality of finely-divided magnetic particles, each of which is coated with a layer directly bonded thereto of first protein molecules specific to the select protein, is dispersed in the liquid sample. The select protein, if present attaches to the protein bonded to the magnetic particles. The magnetic particles are magnetically retrieved, washed and then treated with a cleaving agent solution in direct contact with a metallized surface. The select protein, if present, detaches from the protein-coated magnetic particles and attaches to the metallized surface, which is examined for presence of the select protein. The magnetic particles which are said to be useful are those useful in U.S. Pat. No. 3,970,518 and the size range for the particles is again colloidal to about 10 microns. In the only example use is made of nickel particles about 1 micron (10.sup.4 .ANG.) in diameter. Synthesis of these particles is difficult and the particles have been found to have a tendency to aggregation during protein coupling and cell labeling procedures.
U.S. Pat. No. 4,230,685 of Senyei et al. is concerned with magnetic separation of cells and the like and with microspheres for use therein. It discusses the teaching of U.S. Pat. No. 3,970,518 and says that there is no literature verification that uncoated magnetic particles can be made to bind effectively with antibodies. It refers to published procedures in which particles of magnetic material are contained in microspheres formed from polymers which can be coupled to antibodies. Mention is made of magnetically responsive microspheres formed from acrylate polymer, such as hydroxyethyl methacrylate, or polyacrylamide-agarose microspheres. Such microspheres can be chemically coupled to antibodies with glutaraldehyde or other di-aldehyde. One described procedure involves the chemical attachment of diaminoheptane spacer groups to the microspheres, which are then chemically linked to the antibodies by the glutaraldehyde reaction. Senyei et al state that although effective bonding of the antibodies can be obtained, such procedures are difficult since aggregation of microspheres can readily occur and the preparative procedure is time consuming. Further, random attachment of the antibodies to the magnetic particle means that that portion of the antibody which binds to the antigen, the Fab region, may not be available for binding. Senyei et al. propose to overcome these various disadvantages by using magnetically responsive microspheres having staphylococcal Protein A associated with the surfaces thereof. It is known that staphylococcal Protein A selectively binds to antibodies through the Fc region of the antibodies which is remote from the Fab region. Consequently the antibodies are arranged in oriented attachment with the Fab arms of the antibodies extending outwards. To attach the staphylococcal Protein A to the magnetic microspheres use is made of a polymer matrix material which does not mask the antibody binding sites of Protein A. The preferred matrix material is albumin but other materials mentioned are other amino acid polymers and synthetic polymers such as acrylate polymers. Examples mentioned are methyl methacrylate, hydroxyethyl methacrylate, methacrylic acid, ethylene glycol dimethacrylate, agarose polymers, polyacrylamide polymers or mixtures of such polymers. Albumin is the only polymer matrix material whose use is demonstrated in a working example. According to column 4 lines 24 to 27, the microspheres of Senyei et al. range in size from 0.2 to 100 microns (2000 to 10.sup.6 .ANG.) in diameter preferably from about 0.5 to 2.0 microns (5000 to 2.times. 10.sup.4 .ANG.).